This is a revised competitive renewal grant application (R01NS48216-A1) to study the role of Na+/H+ exchanger isoform 1 (NHE1) in focal ischemic damage. Limited information is known about the role of NHE-1 in cerebral ischemia and whether inhibition of NHE-1 is neuroprotective against ischemic brain damage. In the initial funding period, we found that NHE-1 was essential in regulation of somata pHi in cortical astrocytes and neurons. NHE-1 activity in astrocytes and neurons was stimulated during reoxygenation (REOX) following oxygen and glucose deprivation (OGD). Either pharmacological inhibition or genetic ablation of NHE-1 activity protected cells from ischemic damage in in vitro and in vivo ischemic models. Excessive stimulation of NHE-1 activity led to dysregulation of intracellular Na+ and Ca2+ homeostasis in conjunction of reversal of Na+/Ca2+ exchange (NCXrev). Many important issues remain unresolved. First, the postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury. Dendritic beading and injury are an early hallmark of neuronal injury in the absence of neuronal death. Na+ influx and mitochondrial dysfunction are important in the acute dendritic injury. However, it is unknown whether NHE-1 and NCXrev contribute to the selective vulnerability of postsynaptic neuronal dendrites. Secondly, activated microglia produce a respiratory burst via NADPH oxidase. Therefore, activation of microglia is associated with a large amount of intracellular H+ generation. But, the role of NHE-1 in regulation of microglial pHi and inflammatory responses following ischemia remains unexplored. In the next stage of our study, we will propose two hypotheses: 1) the robust activity of Na+- dependent H+ extrusion mechanism (NHE) in conjunction with activation of NCXrev contributes to ischemic dendritic injury by excessive accumulation of Na+, Ca2+, and mitochondrial dysfunction; 2) NHE-1 activity is stimulated upon microglia activation to meet the demand of maintaining the optimal pHi for NADPH oxidase function following ischemia. Moreover, NHE1-mediated [Na+]i overload and subsequent activation of NCXrev may elevate [Ca2+]i and enhance the p38 MAPK- and/or NF-B-mediated inflammatory responses. Therefore, inhibition of NHE-1 activity pharmacologically or via genetic ablation may offer neuroprotection against the acute cerebral ischemic injury via blocking these cellular events. The hypotheses will be tested in three Aims. The results of the proposed studies will enhance our understanding of the appeared paradoxical role of NHE1 in the CNS following ischemia. This knowledge will be beneficial for developing a more effective approach to stroke treatment.